Subtilisin-A was colyophilized with various types of support materials, such as Amberlite IRC-50, Celite545, chitosan, DEAE-cellulose, DOWEX-1, zeolite, glass bead, and polystyrene. The colyophilized enzyme was used for the optical resolution of racemic l-phenylethylamine with 2,2,2-trifluoroethylbutyrate in 3-methyl-3-pentanol. The enzyme activity in organic media changed dramatically according to the hydration state of the support materials before lyophilization. This effect was especially marked with supports of high water capacity (aquaphilicity), such as chitosan and DEAE-cellulose. By hydrating these supports of high aquaphilicity prior to lyophilization, subtilisin-A activity in organic media increased ca. 4-8 times, depending upon the supports used. This result suggests that the hydration state of aquaphilic support materials for colyophilization is critical to determining enzyme activity in organic solvents.